ABSTRACT Anti-resorptive medications have helped millions of patients suffering from bone-wasting conditions. However, a side effect called medication-related osteonecrosis of the jaw has caused significant anxiety towards these otherwise useful medications, causing a sharp decline in prescriptions, despite the high benefit to risk ratio. Unlike other anti-resorptive medications, bisphosphonates (including zoledronate) accumulate in high concentration in the bone matrix of alveolar bone following the sessation of systemic treatment. Our preliminary results show that matrix-bound zoledronate contributed to the exclusivity of osteonecrosis to the jawbone (bisphosphonate-related osteonecrosis of the jaw; BRONJ), but was not the only factor. In human patients, certain oral bacteria were common in BRONJ lesions (for example, Fusobacterium nucleatum and Actinomyces israelii), suggesting that these bacteria may also be important for the pathogenesis of BRONJ. Our central hypothesis is that matrix-bound bisphosphonates render the oral microenvironment favorable to bacterial colonization of alveolar bone, shifting the balance of the bone regenerative response towards bone destruction rather than effective regeneration, even after stopping the systemic bisphosphonate treatment. In this proposal, we will study the interaction between predominant BRONJ-associated oral bacteria (F. nucleatum and A. israelii) with systemic and matrix-bound bisphosphonates during the pathogenesis of BRONJ. Specific Aim 1 will test whether matrix-bound ZA enhances attachment of predominant BRONJ-associated bacteria to mineralized matrix. We will examine the effects of matrix-bound ZA on the attachment mechanisms of two bacterial strains most commonly identified in necrotic alveolar bone (F. nucleatum and A. israelii), whether the effects can be reversed by chelating matrix-bound ZA using EDTA, and the effect of F. nucleatum and A. israelii on osteoclast differentiation, function, and cross-talk with osteoblast and endothelial cells with and without ZA treatment. Specific aim 2 will test whether BRONJ-associated bacteria are critical for the development of BRONJ. We will test the hypothesis that matrix-bound zoledronate render the oral microenvironment favorable to bacterial colonization, even after stopping systemic bisphosphonate treatment. Such bacterial colonization (F. nucleatum and A. israelii) of zoledronate-treated animals will induce osteonecrosis following invasive surgical procedures at extra-oral bone sites, an effect that can be ameliorated by local removal of zoledronate using EDTA. This project is based on the specific aims accomplished during the first R15 award and is an extension of the same line of research. The award also provided the sole support for 11 graduate, pre-doctoral, and undergraduate students. Successful renewal will allow the continuation of student research at the PI?s lab. 11